Nomuraeae rileyi-origin ecdysteroid 22-oxidase and molt hormone inactivation system with the use of the same

ABSTRACT

A material and a system are provided that efficiently inactivate a molting hormone by means of a protein having ecdysteroid 22-oxidase activity.

TECHNICAL FIELD

The present invention relates to an ecdysteroid 22-oxidase that is isolated from Nomuraea rileyi, which is an entomopathogenic fungus, and a molting hormone inactivation system using same.

BACKGROUND ART

It is known that molting of arthropods, including insects and crustaceans, is induced by several types of ecdysteroids having molting hormone activity.

At least two uses have been developed for these molting hormones.

One of these is the application thereof to growth control, including acceleration of the timing of molting or metamorphosis of individuals and equalization of pupation. This enables, for example, silk thread production in silkworms to be controlled.

The other use is the application thereof to a gene expression system in a cultured cell line, a transgenic animal, or a transgenic plant, the system enabling a high level of expression of a target gene and control of expression timing to be obtained by a molting hormone treatment. This is based on the finding that the molting hormone binds to a molting hormone receptor, which is a transcription factor, and further binds to a molting hormone responsive element on a molting hormone responsive gene, thus controlling the transcription activity of the responsive gene.

For example, a molting hormone receptor and a target gene having a molting hormone responsive element incorporated into its transcription control region are first introduced into these systems, and the intracellular molting hormone concentration is increased by using a method such as addition to a cultured cell line (Christopherson, K. S. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6314–6318), injection into an animal (No, D et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3346–3351), or absorption via a plant root (Martinez, A. et al. (1999) The Plant Journal 19, 97–106), thus inducing expression of a target gene product. Among these methods, one employing a cultured cell line has already been put into practice as a kit.

On the other hand, a technique for enhancing the activity of the molting hormone without using the molting hormone itself has also been developed. For example, examples thereof include the application to insect pest control of an ecdysteroid having a high molting hormone activity, and a more stable and strong molting hormone agonist having no ecdysteroid skeleton.

In this way, techniques for increasing molting hormone activity have already been developed.

In contrast, hardly any techniques for decreasing molting hormone activity, that is, techniques for inactivating the molting hormone present within a body or cells, have been developed.

A baculovirus-derived ecdysteroid UDP-glucosyltransferase gene (JP, A, 11-123079) has recently been receiving attention as a gene of an enzyme having an ability to inactivate the molting hormone. However, since there are defects such as it being necessary for UDP-glucose to be present at the same time in order for the enzyme to function, it has not yet been put into practice as a purified enzyme preparation or a recombinant protein.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a material and a system that can inactivate the molting hormone efficiently.

In carrying out an intensive investigation in order to achieve this object while recognizing that inactivation of the molting hormone is extremely important in applications such as growth control of insects, etc. and control of induced expression of a target gene product, the present inventors have noted that all ecdysteroids having molting hormone activity have a hydroxyl group at the 22-position, and when this position is modified, the molting hormone activity is markedly degraded. More specifically, it has been found that the above-mentioned object can be attained by isolating an ecdysteroid 22-oxidase from Nomuraea rileyi, which is an entomopathogenic fungus, and oxidizing the 22-hydroxyl group of an ecdysteroid into a keto group with this enzyme or a modified protein thereof, and the present invention has thus been accomplished.

That is, the present invention relates to protein (a) or (b) below:

(a) a protein having the amino acid sequence shown by Sequence Listing SEQ ID NO:2, or

(b) a protein having an amino acid sequence in which one or a plurality of amino acids are deleted, substituted, or added in Sequence Listing SEQ ID NO:2, the protein having ecdysteroid 22-oxidase activity.

Furthermore, the present invention relates to a gene having DNA (a) or (b) below:

(a) a DNA having the base sequence shown by Sequence Listing SEQ ID NO:1, or

(b) a DNA that codes for a protein having ecdysteroid 22-oxidase activity and that hybridizes under stringent conditions with the DNA having the base sequence (a).

Moreover, the present invention relates to a method for inactivating the molting hormone of an arthropod by administering the protein to the arthropod.

Furthermore, the present invention relates to a method for controlling growth of an arthropod by inactivating the molting hormone using the protein.

Moreover, the present invention relates to a method for controlling growth of an insect by inactivating the molting hormone using the protein.

Furthermore, the present invention relates to a method for producing silk thread, the method comprising administering the protein to a silkworm so as to control the diameter of thread spun by the silkworm.

Moreover, the present invention relates to a method for suppressing expression of a molting hormone-inducible gene, the method comprising administering the protein to a transformant.

The protein according to the present invention is an enzyme having activity in oxidizing the 22-hydroxyl group of the molting hormone and inactivating it. Administering this enzyme internally to an arthropod therefore inactivates its molting hormone and enables its growth to be controlled.

The enzyme according to the present invention can be used for controlling the growth of insects including silkworms. In particular, by administering the enzyme according to the present invention to the silkworm, silk thread having high product value due to it being finer than normal silk thread can be produced.

The enzyme according to the present invention can be used for controlling not only the growth of insects but also the growth of crustaceans that use an ecdysteroid as the molting hormone in the same manner as insects.

Furthermore, in order to obtain the enzyme, a system can be employed in which a silkworm is infected with a recombinant baculovirus into which has been incorporated a gene according to the present invention having the base sequence shown by Sequence Listing 2, and a large amount of the protein is expressed in its blood and is collected.

Use of the enzyme or the gene according to the present invention, in a system that induces expression of a target gene product by increasing the intracellular molting hormone concentration by the addition, injection, or root absorption of the molting hormone, enables expression of the target gene to be stopped at will. Therefore, in transformants such as cultured cells, transgenic animals, and transgenic plants, the gene expression system using the molting hormone can be controlled in a negative direction, and this enables the applications of gene expression systems using transformants to be extended.

The present invention is explained in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A shows the structures of ecdysteroids found in insect bodies, and B shows HPLC charts of ecdysteroids and products of oxidation by an enzyme according to the present invention.

FIG. 2: diagram showing the oxidation activity of the ecdysteroid 22-oxidase expressed by the cDNA according to the present invention.

FIG. 3: photographic diagram showing the result of a Western blot analysis using a polyclonal antibody to a purified enzyme according to the present invention.

FIG. 4: A and B are photographic diagrams showing the effects of the enzyme according to the present invention respectively on fourth instar larvae (penultimate instar) and fifth instar larvae of silkworms.

FIG. 5: diagrams showing the accumulation of a modified product in which the 22-hydroxyl group has been oxidized within the bodies of fourth instar larvae (A) and fifth instar larva (B) of silkworms injected with an enzyme solution and a comparison with non-treated fourth instar larvae (C) and non-treated fifth instar larvae (D).

FIG. 6: image analyzer (GS-250, Bio-Rad) showing that 20-hydroxy-22-dehydroecdysone whose 22-position has been modified by the enzyme according to the present invention has no effect in inducing the expression of EcR mRNA.

BEST MODE FOR CARRYING OUT THE INVENTION

Sequence Listing 1 shows the cDNA of ecdysteroid 22-oxidase isolated from Nomuraea rileyi according to the present invention and an amino acid sequence predicted therefrom. Furthermore, Sequence Listing 2 shows a predicted amino acid sequence of the ecdysteroid 22-oxidase.

With regard to cloning of cDNA, a protein is purified from a Nomuraea rileyi culture solution using as an indicator the activity for ketonization (oxidation) of the 22-hydroxyl group of ecdysone, which is one type of the molting hormone, and N-terminal and internal amino acid sequences are determined. Moreover, the entire length of the cDNA is cloned by PCR based on these amino acid sequences.

FIG. 1A shows the structures of ecdysteroids found in insect bodies and the products of their oxidation by the enzyme according to the present invention. As shown in FIG. 1B, these ecdysteroids can be easily detected by HPLC. That is, a reverse phase column (TSKgel ODS-80Ts, 4.6 mm×150 mm TOSO) was mounted in an HPLC (LC 10-AT, Shimadzu), and the absorbance at 245 nM was measured by a standard UV detector. In this procedure, the flow rate was 0.6 ml/min, the column retention temperature was 40° C., and a sample was passed in an acetonitrile gradient (20–30%) for 40 minutes.

When a predicted coding region (A₁₁₁-T₁₈₉₃ in Sequence Listing 1) of cDNA according to the present invention was expressed in SF-9 cells using a protein expression system (BAC-TO-BAC Baculovirus Expression Systems, Gibco BRL) employing a baculovirus, strong ecdysteroid oxidation activity was observed in the culture solution on the 7th day after starting culturing (FIG. 2). In a Western blot analysis using a polyclonal antibody to the purified present enzyme, it was also confirmed that ecdysteroid 22-oxidase was secreted in the culture solution (FIG. 3).

It is known to a person skilled in the art that, in general, with regard to an amino acid sequence coding for a protein having bioactivity, even when one or a plurality of amino acids are added, deleted, or substituted, the bioactivity can be maintained in some cases. The present invention includes a DNA fragment coding for a protein modified in this manner and having an ecdysteroid 22-oxidase activity. That is, the scope of the present invention also includes a DNA coding for a protein comprising an amino acid sequence of Sequence Listing SEQ ID NO:2 in which one or a plurality of amino acids are added, deleted, substituted, or inserted, the protein having ecdysteroid 22-oxidase activity.

The ‘one or a plurality of’ referred to here usually means 2 to 20, and preferably on the order of 2 to 15, although it depends on the location of the amino acid residue in the tertiary structure of the ecdysteroid 22-oxidase protein at which an amino acid is added, deleted, or substituted, or on the type of the amino acid residue.

A DNA modified in this manner can be obtained by modifying the base sequence of the DNA of the present invention so that a specified amino acid can be deleted, substituted, or added by, for example, a site-directed mutation method.

A modified DNA can also be obtained by subjecting the DNA of the present invention or cells having same to mutation and selecting, from the DNA or the cells, a DNA that hybridizes under stringent conditions with a DNA having the base sequence described by, for example, SEQ ID NO:1 in the Sequence Listing.

The ‘stringent conditions’ referred to here means conditions under which a specific hybrid is formed but no non-specific hybrid is formed. Although it is difficult to express these conditions numerically, the conditions are, for example, those under which nucleic acids having a high homology of at least 99.5% can hybridize, but DNAs having a lower homology than this cannot hybridize.

The present invention is explained below by reference to examples, but the present invention is not limited to the examples below.

EXAMPLE 1 Isolation of Ecdysteroid 22-oxidase from Nomuraea rileyi

Nomuraea rileyi was cultured for 9 days in a liquid culture medium containing an extract from silkworm larvae, the liquid culture (containing the target enzyme) was passed through a 0.45 μm filter, and then stored at 4° C. After collecting a sufficient amount of liquid culture, the enzyme was isolated by an extraction that involved the following 4 steps.

-   1. Precipitation by 50% ammonium sulfate. -   2. Phenyl hydrophobic chromatography (using phenyl-Sepharose) -   3. Gel filtration (using Superdex 200 pg) -   4. Anionic chromatography (using HiTrap Q)

2 to 3 were carried out using HPLC (Model Bio-HPLC system, Tosoh).

In each process, an enzyme reaction was carried out using as a substrate ecdysone, which is described in FIG. 1A, and a fraction containing the target enzyme was determined by monitoring an oxidized product using HPLC. After final purification, SDS PAGE was carried out, and it was confirmed by silver staining that a single protein was obtained by the purification.

EXAMPLE 2 Determination of Ecdysteroid 22-oxidase Gene Sequence

The N-terminal sequence of the enzyme isolated according to the method described in Example 1 was first analyzed by an amino acid sequencer to determine the N-terminal amino acid sequence. Furthermore, the N-terminal sequence of a decomposition product obtained by partial decomposition of the enzyme preparation by V8 protease was analyzed by an amino acid sequencer to determine the internal amino acid sequence of this enzyme.

Based on the N-terminal sequence and the internal sequence of the enzyme thus determined, four types of degenerate primers were designed, that is, as forward primers, E22o.6 primer (SEQ ID NO:3; coding for the amino acid sequence LPQGGCR (21 to 27)) and E22o.2 primer (SEQ ID NO:4; coding for the amino acid sequence CRCIPGE (26 to 32)) and, as reverse primers, Int.R1 primer (SEQ ID NO:5; reverse coding for the amino acid sequence QNVNNAW (74 to 80)) and Int.R2 primer (SEQ ID NO:6; reverse coding for the amino acid sequence DQGQNVN (71 to 77)). A partial cDNA of this enzyme was cloned by RT-PCR using these primers and employing mRNA extracted from cultured Nomuraea rileyi as a template.

PCR was carried out twice with different primer sets. That is, the E22o.6 primer and the Int.R1 primer were used in the first reaction, and the E22o.2 primer and the Int.R2 primer were used in the second reaction. Finally, the 115 base sequence from 206 to 320 in the molting hormone oxidase cDNA (entire length: 1963 bases) was amplified, and the base sequence was determined.

By designing primers in the partial cDNA thus cloned, and further carrying out 5′RACE and 3′RACE, which are types of modified RT-PCR methods, using the SMART RACE cDNA Amplification Kit (manufactured by CLONTECH), a cDNA covering the entire length of the mRNA of this enzyme was cloned. In the 3′RACE a region from 209 to 1963 of the entire base sequence was amplified using E22o.RF1 primer (SEQ ID NO:7; corresponding to 209 to 231 of the entire base sequence of E22o) and its base sequence was determined. In the 5′RACE, a region from 1 to 290 of the entire base sequence was amplified using E22o.RR1 primer (SEQ ID NO:8; corresponding to the reverse chain of the entire base sequence of E22o) and its base sequence was determined.

By superimposing regions cloned as above by RT-PCR, 5′RACE, and 3′RACE, the entire length of the cDNA of the molting hormone oxidase of Nomuraea rileyi was determined.

EXAMPLE 3 Effect of Ecdysteroid 22-oxidase on Silkworm Larvae

Fourth instar and fifth instar silkworm larvae were injected with an enzyme solution containing the enzyme according to the present invention at 1.6 units/20 μl/head, and the growth thereafter was examined. ‘1 unit’ represents the enzyme activity that can oxidize 1 nM of ecdysone in 1 minute.

Fourth instar (penultimate instar) silkworm larvae usually molt into fifth instar (final instar) larvae approximately on Day 5, and fifth instar larvae start to form a cocoon approximately on Day 7 and pupate on the 11th day. However, when the present enzyme was injected into the body of a silkworm at the beginning of the fourth instar, it started to form a cocoon approximately 7 days after the injection and pupated 11 days later (FIG. 4A). On the other hand, when it was injected on the 7th day of the fifth instar, it remained in the larval stage for at least 10 days after the injection, and finally died without pupating (FIG. 4B).

In each case, when the ecdysteroid in the blood was examined after injecting the enzyme solution, hardly could any 20-hydroxyecdysone, which is an active form of the molting hormone, or its precursor ecdysone be detected, and it was found that a large amount of the modified products having the 22-hydroxyl group oxidized had accumulated (FIG. 5).

In this way, by injecting a silkworm with the enzyme according to the present invention, the molting hormone within the silkworm body is inactivated, and growth control such as precocious metamorphosis, extension of the spinning period, and inhibition of metamorphosis can be controlled according to the timing of the injection.

EXAMPLE 4 Transcription Inducing Effect of 22-oxidized Ecdysteroid on Molting Hormone-Inducible Gene

It is known that transcription of a molting hormone receptor (EcR) gene is induced by the molting hormone itself, which is a ligand of the gene. Various concentrations of 20-hydroxyecdysone or 20-hydroxy-22-dehydroecdysone whose 22-position had been modified by the enzyme according to the present invention were added to cultured silkworm anterior silk gland, and expression of EcR mRNA was examined after several hours. The result was that, in the case where 20-hydroxyecdysone was added, the expression of EcR mRNA increased in line with the amount added, but in the case where 20-hydroxy-22-dehydroecdysone was added, there was no expression inducing effect (FIG. 6). It was thus confirmed that the ecdysteroid whose 22-position had been modified by the enzyme according to the present invention had no transcription inducing activity for the molting hormone-inducible gene.

As hereinbefore described, the ecdysteroid 22-oxidase according to the present invention has a high ability to inactivate the molting hormone, and use of the enzyme enables growth of an insect and expression of a molting hormone-inducible gene to be controlled effectively.

INDUSTRIAL APPLICABILITY

Use of the enzyme according to the present invention enables growth of an insect to be controlled by efficiently inactivating the insect molting hormone. Furthermore, use of the enzyme enables finer silk thread than usual to be produced. Moreover, use of a gene coding for the enzyme enables expression of the gene to be controlled in a system in which expression of a target gene product is induced by increasing the intracellular molting hormone concentration.

Consequently, the present invention can be applied to the silk thread industry and an industry involved in the production of a specific protein such as, for example, the pharmaceutical industry.

[Sequence Listing Free Text]

-   [SEQ ID NO:1] Base sequence of DNA coding for Nomuraea     rileyi-derived ecdysteroid 22-oxidase. -   [SEQ ID NO:2] Amino acid sequence of Nomuraea rileyi-derived     ecdysteroid 22-oxidase. -   [SEQ ID NO:3] Description of artificial sequence: E220.6 primer for     RT-PCR. ‘n’ denotes inosine. -   [SEQ ID NO:4] Description of artificial sequence: E220.2 primer for     RT-PCR. ‘n’ denotes inosine. -   [SEQ ID NO:5] Description of artificial sequence: Int.R1 primer for     RT-PCR. ‘n’ denotes inosine. -   [SEQ ID NO:6] Description of artificial sequence: Int.R2 primer for     RT-PCR. ‘n’ denotes inosine. -   [SEQ ID NO:7] Description of artificial sequence: EE22o.RF1 primer     for modified RT-PCR. -   [SEQ ID NO:8] Artificial sequence: EE22o.RR1 primer for modified     RT-PCR. 

1. An isolated protein selected from: (a) a protein having the amino acid sequence of SEQ ID NO:2, or (b) a modified protein having an amino acid sequence of SEQ ID NO:2 in which one or 2 to 20 amino acids are deleted, substituted, or added, the modified protein having ecdysteroid 22-oxidase activity.
 2. An isolated DNA selected from: (a) a DNA having the nucleotide sequence of SEQ ID NO:1 or (b) a DNA that encodes a protein having ecdysteroid 22-oxidase activity and hybridizes with the DNA having the nucleotide sequence (a) under conditions under which nucleic acids having a homology of at least 99.5% with the nucleotide sequence (a) can hybridize therewith.
 3. A method for producing silk thread, the method comprising administering the protein according to claim 1 to a silkworm so as to control the diameter of silk thread spun by the silkworm; and allowing the silkworm to spin silk thread.
 4. A method for suppressing expression of a gene in a non-human transformant, wherein expression of the gene is induced by increasing the intracellular level of an ecdysteroid in the transformant, the method comprising administering the protein according to claim 1 to the transformant, wherein the gene is inducible by 20-hydroxyecdysone and the transformant is an isolated host sell that has been treated with 20-hydroxyecdysone.
 5. The isolated protein according to claim 1, which is a protein having the amino acid sequence of SEQ ID NO:2.
 6. The isolated DNA according to claim 2, wherein the DNA has the nucleotide sequence of SEQ ID NO:1.
 7. The method according to claim 4, wherein the protein is a protein having the amino acid sequence of SEQ ID NO:2. 